Jean Martin Charcot, Pierre Marie and Howard Henry Tooth characterized Charcot-Marie-Tooth (CMT) in 1886. Today, CMT is the most common inherited disease of the peripheral nervous system, affecting approximately 150,000 Americans. Four major forms of CMT are now recognized (referred to as CMT 1-4). The major differences between forms are the linked gene and the primary cell type affected (Schwann cells versus nerves). Charcot-Marie-Tooth type 2E (CMT2E) is a sub-type of CMT2. CMT2E is an autosomal dominant disorder that affects peripheral nerve axons. A series of recent reports linked 16 mutations in neurofilament light (NF-L) to CMT2E. NF-L, with CMT2E linked mutations, expressed in cell culture disrupts neurofilament organization and transport. Additionally, mutant NF-L functions in a dominant manner in primary neuronal cultures. Interestingly, complete loss of all axonal neurofilaments, through targeted deletion of NF-L in mouse, does not result in overt pathology. Therefore, little is known about the mechanism(s) involved in the pathogenesis of NF-L linked CMT2E. Our objective is to develop two animal models of CMT2E so that we can analyze disease pathogenesis in NF-L linked CMT. We will achieve this by developing two independent lines of gene knock-in mice. One line of mice will express NF-L with proline 8 mutated to arginine, and the other will express NF-L with glutamate 397 mutated to lysine. We will analyze these mice for pathological changes at both the cellular and organism level. At the cellular level, we will look for alterations in neurofilament accumulation and organization in axons, alterations in radial axonal growth and alterations in myelination. We will, also, monitor the mice for the appearance of CMT-like symptoms. Specifically, we will analyze muscle wasting, gait, thermal perception and nerve conduction velocities. Generating two lines of gene-targeted mice will address important questions relevant to therapy development. For example, as our proposed mutations are in distinct functional domains of the protein, do these mutations result in disease through different mechanisms? Do different NF-L mutations result in variable onset or severity of disease? Additionally, we will analyze cellular phenotypes independently in motor and sensory axons to determine if both neuronal types are equally vulnerable to expression of mutant NF-L. The use of a time course will allow us to identify early changes associated with expressing mutant NF-L. Generating and analyzing animal models of CMT2E is the first key step in identifying novel sites and strategies for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Charcot-Marie-Tooth (CMT) is the most common inherited disease of the peripheral nervous system affecting approximately 150,000 Americans with severe cases of CMT2 presenting with respiratory dysfunction in infants and with laryngeal weakness, hoarseness and respiratory difficulties in adults. Mutations in a cytoskeletal protein, neurofilament light (NF-L), have been linked to CMT2E. We are interested in developing animal models of CMT2E to determine how mutations in NF-L lead to the development of disease and to identify novel sites and strategies for therapeutic intervention. [unreadable] [unreadable]